Haoyi, good work. Maybe give the enzyme assay another shot - but I would recommend working on your PCRs first. . - Dr. B 07/05/2017
Week 3&4
Enzyme Assay
Figure 1. Spectrometer graph of enzyme from samples A to H of run 1. Wavelength of interest at 410nm. N=1.
Comments: Bad results that did not make sense. All absorbance values at around 1.8, except A, regardless of amount enzyme added.
Restriction Enzyme Digest
Figure 1. Gel electrophoresis image of L2 (ladder), L3 (uncut plasmid), L4 (EcoRI cut), L5 (PVuII cut), L6 (EcoRI & PvuII cut) under UV light after 100V of 40 minutes run and 130V of 25 minutes run.
Comments: Uncut plasmid not showing up likely due to false dilution and calculation.
FPLC
Figure 1. Absorbance vs. Sample Numbers for FPLC nanodrop results of YopH. YopH expected at around peak of 40, also possible at first peak around 30. N=3 trials.
Comments: YopH expected at peak around 40 because it was about 35kDa and closest to Ovalbumin (~40kDa) in standard. YopH might have clumped together to appear larger, coming out at around sample 30.
YopH Purification
Figure 1. Nanodrop results of elution 1 and elution 2 during YopH purification. #16 to 19 for elution 1, #20 to 24 for elution 2. N=4.
Comments: Nanodrop was not functioning properly, which may contribute to the strange results. Graph showed large amount of DNA contamination, it was unclear where it came from. Elu2 should have lower abs value than Elu1.
YopH Characterization
Figure 1. SDS Page gel electrophoresis results of characterization of YopH, with L1=ladder, L2 to L8 samples 0 to 6 of Haoyi, L9 to L10 samples 5 and 6 of Esther. According to New England biolabs ladder, YopH was at around 32kDa.
Comments: Protein purification was a failure because large amount of protein (and potentially DNA, unknown since it did not show on gel) contamination shown on gel. YopH was obtained according to the especially thick band in sample 6 (Elution 2).
Figure 1. 2 tubes of pGBR22 DNA (S1 and S2) analyzed for DNA concentration in nanodrop. Graph showing nanodrop results of S2 Trial 3. N=3 for S2, N=4 for S1 due to an outlier result of 0.047. Row 2 to 5 for sample 1, Row 6 to 8 for sample 2.
Figure 2. Midiprep samples of pGBR22 before centrifuging. Purple pellet was pGBR22 plasmid showing.
Comments: It was best to combine the 2 tubes of DNA samples to get a higher concentration. The low number showed not much DNA was obtained. None of the other samples using plasmids other than pGBR22 contained this strange yellow color during MidiPrep.
pGBR22 PCR Gel Electrophoresis
Figure 1. Reverse color image of DNA gel electrophoresis of PCR samples A, B, C, D containing pGBR22 plasmid sequence in different dilutions of A (1:1000), B(1:1000), C (1:100), D (0). Gel ran for 45 minutes. Gel image taken in X ray, black bands indicating DNA. L1 was ladder, L2 to 5 were A,B,C,D of Haoyi, L6 to 9 were that of Sung-Hoon.
Comments: L2 to 5 gave incorrect results, but L6 to 9 did. It was probably due to a mix up of samples.
Primer Dilutions
Figure 1. Information sheet on diluted primer.
DNA sequencing
Figure 1. DNA sequencing of pGBR22, chromatogram.
Haoyi, good work. Maybe give the enzyme assay another shot - but I would recommend working on your PCRs first. . - Dr. B 07/05/2017
Week 3&4
Enzyme Assay
Figure 1. Spectrometer graph of enzyme from samples A to H of run 1. Wavelength of interest at 410nm. N=1.
Comments: Bad results that did not make sense. All absorbance values at around 1.8, except A, regardless of amount enzyme added.
Restriction Enzyme Digest
Figure 1. Gel electrophoresis image of L2 (ladder), L3 (uncut plasmid), L4 (EcoRI cut), L5 (PVuII cut), L6 (EcoRI & PvuII cut) under UV light after 100V of 40 minutes run and 130V of 25 minutes run.
Comments: Uncut plasmid not showing up likely due to false dilution and calculation.
FPLC
Figure 1. Absorbance vs. Sample Numbers for FPLC nanodrop results of YopH. YopH expected at around peak of 40, also possible at first peak around 30. N=3 trials.
Comments: YopH expected at peak around 40 because it was about 35kDa and closest to Ovalbumin (~40kDa) in standard. YopH might have clumped together to appear larger, coming out at around sample 30.
YopH Purification
Figure 1. Nanodrop results of elution 1 and elution 2 during YopH purification. #16 to 19 for elution 1, #20 to 24 for elution 2. N=4.
Comments: Nanodrop was not functioning properly, which may contribute to the strange results. Graph showed large amount of DNA contamination, it was unclear where it came from. Elu2 should have lower abs value than Elu1.
YopH Characterization
Figure 1. SDS Page gel electrophoresis results of characterization of YopH, with L1=ladder, L2 to L8 samples 0 to 6 of Haoyi, L9 to L10 samples 5 and 6 of Esther. According to New England biolabs ladder, YopH was at around 32kDa.
Comments: Protein purification was a failure because large amount of protein (and potentially DNA, unknown since it did not show on gel) contamination shown on gel. YopH was obtained according to the especially thick band in sample 6 (Elution 2).
DNA sequencing
Figure 1. pGBR22 reverse, chromatogram.
Figure 2. pGBR22 forward, chromatogram.
Week 1&2
Midiprep
Figure 1. 2 tubes of pGBR22 DNA (S1 and S2) analyzed for DNA concentration in nanodrop. Graph showing nanodrop results of S2 Trial 3. N=3 for S2, N=4 for S1 due to an outlier result of 0.047. Row 2 to 5 for sample 1, Row 6 to 8 for sample 2.
Figure 2. Midiprep samples of pGBR22 before centrifuging. Purple pellet was pGBR22 plasmid showing.
Comments: It was best to combine the 2 tubes of DNA samples to get a higher concentration. The low number showed not much DNA was obtained. None of the other samples using plasmids other than pGBR22 contained this strange yellow color during MidiPrep.
pGBR22 PCR Gel Electrophoresis
Figure 1. Reverse color image of DNA gel electrophoresis of PCR samples A, B, C, D containing pGBR22 plasmid sequence in different dilutions of A (1:1000), B(1:1000), C (1:100), D (0). Gel ran for 45 minutes. Gel image taken in X ray, black bands indicating DNA. L1 was ladder, L2 to 5 were A,B,C,D of Haoyi, L6 to 9 were that of Sung-Hoon.
Comments: L2 to 5 gave incorrect results, but L6 to 9 did. It was probably due to a mix up of samples.
Primer Dilutions
Figure 1. Information sheet on diluted primer.
DNA sequencing
Figure 1. DNA sequencing of pGBR22, chromatogram.